Dendritic macroporous hydrogels prepared by crystal templating

ABSTRACT

The present invention includes a hydrogel and a method of making a porous hydrogel by preparing an aqueous mixture of an uncrosslinked polymer and a crystallizable molecule; casting the mixture into a vessel; allowing the cast mixture to dry to form an amorphous hydrogel film; seeding the cast mixture with a seed crystal of the crystallizable molecule; growing the crystallizable molecule into a crystal structure within the uncrosslinked polymer; crosslinking the polymer around the crystal structure under conditions in which the crystal structure within the crosslinked polymer is maintained; and dissolving the crystals within the crosslinked polymer to form the porous hydrogel.

This application is a continuation of U.S. patent application Ser. No.14/277,170, filed May 14, 2014 and entitled “DENDRITIC MACROPOROUSHYDROGELS PREPARED BY CRYSTAL TEMPLATING”, which is a continuation ofU.S. patent application Ser. No. 13/909,707, filed Jun. 4, 2013 andentitled “DENDRITIC MACROPOROUS HYDROGELS PREPARED BY CRYSTALTEMPLATING”, now U.S. Pat. No. 8,728,499, issued May 20, 2014, which isa continuation of U.S. patent application Ser. No. 12/919,667, filedAug. 26, 2010 and entitled “DENDRITIC MACROPOROUS HYDROGELS PREPARED BYCRYSTAL TEMPLATING”, now U.S. Pat. No. 8,668,863, issued Mar. 11, 2014,which claims priority to Patent Cooperation Treaty Application SerialNo. PCT/US2009/035257, filed Feb. 26, 2009 and entitled “DENDRITICMACROPOROUS HYDROGELS PREPARED BY CRYSTAL TEMPLATING”, which claimspriority to U.S. Provisional Patent Application Ser. No. 61/031,651,filed Feb. 26, 2008 and entitled “DENDRITIC MACROPOROUS HYDROGELSPREPARED BY CRYSTAL TEMPLATING.” The content of each of the aboveapplications is hereby incorporated by reference.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grant no.BES0201744 and BES0500969 awarded by the National Science Foundation.The government has certain rights in this invention.

BACKGROUND

The present invention relates in general to the field of hydrogels, andmore particularly, to dendritic macroporous hydrogels prepared bycrystal templating.

Without limiting the scope of the invention, its background is describedin connection with hydrogels. Hydrogels are generally polymer chainnetworks that are water-insoluble, but that absorb water. Oftendescribed as being “superabsorbent,” hydrogels are able to retain up to99% water and can be made from natural or synthetic polymers. Often,hydrogels will have a high degree of flexibility due to their high watercontent. Common uses for hydrogels include: sustained drug release, asscaffolds (e.g., in tissue engineering), as a thickening agent, as abiocompatible polymer, in biosensors and electrodes and for tissuereplacement applications. Natural hydrogels may be made from agarose,methylcellulose, hyaluronic acid (HA), and other naturally-derivedpolymers.

One method for making hydrogels is a taught by U.S. Pat. No. 7,307,132,issued to Nestler, et al., for a method of producing low-odorhydrogel-forming polymers. Briefly, a low-odor hydrogel-forming acrylicacid polymer is prepared by preparing a polymeric hydrogel byfree-radically polymerizing a monomer composition comprising at least50% by weight of acrylic acid in an aqueous polymerization medium andconverting said hydrogel into a particulate hydrogel or intohydrogel-forming powder; and optionally treating the particulatehydrogel or said hydrogel-forming powder with a crosslinking substancewhich, actually or latently, contain at least two functional groupscapable of reacting with the carboxyl groups on the addition polymer;characterized by the acrylic acid used in step (a) containing less than400 ppm of acetic acid and propionic acid.

Another method is taught by U.S. Pat. No. 6,943,206, issued to Haraguchifor an organic/inorganic hybrid hydrogel and method for manufacturing.Briefly, an organic/inorganic hybrid hydrogel is said to have superiorhomogeneity, transparency, mechanical properties, and swelling andshrinking properties. A dry body of the organic/inorganic hybridhydrogel is obtained by removing water from said hydrogel. Theorganic/inorganic hybrid hydrogel comprises a water soluble polymer (A),a water swelling clay mineral (B) which can be homogeneously dispersedin water, and water (C), and water (C) is included in athree-dimensional network formed by (A) and (B).

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 shows the chemical structures of biopolymers used in thesehydrogels and a schematic of the crystal-templating technique;

FIGS. 2A to 2F are polarized light (FIGS. 2A and 2D) and phase contrast(FIGS. 2B, 2C, 2E, and 2F) microscopy images of crystal templated HAhydrogel; FIG. 2A) polarized light microscopy image of uncrosslinked HAhydrogel-urea crystal composite; (FIG. 2B) phase contrast microscopyimage of uncrosslinked HA hydrogel-urea crystal composite; (FIG. 2C)phase contrast microscopy image of crosslinked HA hydrogel after removalof the urea crystals; (FIG. 2D) polarized light microscopy image ofuncrosslinked HA hydrogel-urea crystal composite; (FIG. 2E) phasecontrast microscopy image of uncrosslinked HA hydrogel-urea crystalcomposite; and (FIG. 2F) phase contrast microscopy image of crosslinkedHA hydrogel after removal of the urea crystals;

FIGS. 3A to 3C are high magnification images of urea crystals andtemplated HA hydrogels; (FIG. 3A) time lapse series of the urea crystalfront captures with video Brightfield microscopy; (FIG. 3B) atomic forcemicroscopy (AFM) image of the surface of a rinsed urea-templated HAhydrogel in air; and (FIG. 3C) profile of the AFM image perpendicular tothe orientation of the ridges;

FIGS. 4A to 4C show the urea seed crystal technique used to control themorphology of crystals and pores within HA hydrogels; (FIG. 4A) threeseparate nucleation points producing a hydrogel with three radialpatterns; (FIG. 4B) line nucleation points producing a hydrogel withlinear alignment of crystals; and (FIG. 4C) magnified view of FIG. 4B.Scale bars are 1000 μm (FIGS. 4A and 4B) and 100 μm (FIG. 4C);

FIGS. 5A to 5F shows the process of urea-templating scaled up fromdroplets to thick films; (FIG. 5A) photograph of scaled-up free standingfilms of plain and urea-templated HA and alginate; (FIG. 5B) top downview of the scanning electron microscopy images (SEM) of crystaltemplated HA after cross-linking and rinsing; (FIG. 5C) portion of thefilm curled back to expose crevices in the hydrogel created by ureacrystals; (FIG. 5D) cross-section of the hydrogel perpendicular to thepore orientation; (FIG. 5E) parallel cross-section depicting thefibrillar morphology; and (FIG. 5F) magnified view of image of FIG. 5E;and

FIGS. 6A to 6F depict dendritic crystal growth within polymer solutionsdemonstrating many possible combinations of crystallizable molecules andpolymers. The crystals were imaged by polarized light (FIGS. 6A to 6E)and phase contrast microscopy (FIG. 6F): (FIG. 6A) urea crystal andalginate; (FIG. 6B) urea crystal and 4-arm-PEG acrylate; (FIG. 6C) ureacrystal and chitosan; (FIG. 6D) potassium phosphate crystal andhyaluronic acid; (FIG. 6E) b-cyclodextrin crystal and alginate; and(FIG. 6F) magnified image of b-cyclodextrin crystals and alginate. Scalebars are 1000 μm (FIGS. 6A to 6E) and 100 μm (FIG. 6F).

DETAILED DESCRIPTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

The creation of macroporosity within tissue engineering scaffolds isimportant because it influences cellular infiltration, scaffoldremodeling, nutrient diffusion, vascular in-growth and functionalintegration with native tissues. The present inventors have developed anovel crystal-templating technique to fabricate hydrogels withcontinuous dendritic porous networks. No other current method is capableof creating such scaffolds. Additional advantages of this technique arethat it is compatible with natural biomaterials, it is fast, can bescaled up, and does not require any expensive equipment or reagents.Crystal-templating works by growing a dendritic crystal template withina solution of uncrosslinked biopolymer, crosslinking the biopolymeraround the template, and removing the crystals by washing with water.The result is a macroporous hydrogel with a network of pores matchingthe shape of the crystal template. We have used urea crystal templatesto pattern photocrosslinked HA and calcium-crosslinked alginatehydrogels. The crystal-templating technique and the materials created byit address the challenge of fabricating materials with intricatedendritic micro-architecture that is observed in native tissues.

The goal of tissue engineering is to create materials that can replaceor repair injured tissue. To that end it is desirable to have tissueengineered constructs that mimic the microarchitecture of nativetissues. An important structural feature of many tissues is highlybranched networks of vessels and ducts. Examples are the bronchioles,the microvasculature, lymphatic vessels, the ductal networks of salivarygland, mammary gland, and kidney, and the dendritic trees produced byneurons [1-4]. Such networks exhibit branching, multiple length scales,a directional orientation, and three-dimensionality. A tissue maycontain multiple entwined networks; for example, bronchioles, arterialvessels, and venous vessels within the lungs. Although many techniquesare available for creating porosity within tissue engineered constructsthey fall short of reproducing even one such network [5].

Briefly, current techniques available for creating porosity aregas-foaming, lyophilization, thermally-induced phase separation andporogen leaching of salts and uncrosslinked polymer microspheres [6-8].Soft replica molding can transfer patterns from etched silicon topolymers such as poly(lactide-co-glycolide), however this is atwo-dimensional technique and the stacked layers are a poorapproximation of three-dimensional porosity [9-10]. The advantages ofthese methods are that they are easy to implement and scale-up. Thedisadvantage is that they provide little control over pore morphologyand limited compatibility with natural materials. Rapid prototypingtechniques permit precise control over pore morphology through the useof computer-aided design systems [11-13]. Rapid prototyping is suitablefor creation of square and hexagonal lattices but not for the complexthree-dimensional dendritic patterns observed in native tissues.Although rapid prototyping techniques are compatible with syntheticpolymers they are not as suitable for natural materials which are watersoluble and viscous. A further limitation is that the resolution of suchtechniques is larger than the length scale of individual cells andneuronal processes. Scaffolds depicted in the literature typically havefeatures hundreds of microns in length which are too large to preciselyguide cellular infiltration. Fabrication of three-dimensional multilayerconstructs can be impractical because each scaffold must be built layerby layer with costly equipment.

Hydrogel scaffolds are uniquely suited for tissue engineeringapplications because they more closely resemble natural tissues withrespect to mechanical properties, porosity, and water content than doother materials [14]. In particular, polysaccharides, such as hyaluronicacid (HA) and sodium alginate (SA) are attractive materials because theyare composed of the same chemical constituents as native extracellularmatrix components. They also exhibit excellent biocompatibility andnon-immunogenicity. HA has found use as a dermal filler, adhesionbarrier, intra-articular viscosupplement, vitreal substitute, drugdelivery matrix and tissue engineering scaffold for cartilage, skin,adipose and vocal cord [15-21]. Alginate has been used extensively forcell encapsulation, drug delivery and tissue engineering of adipose andcartilage [22-24].

In comparison to synthetic materials, these polysaccharides areconsiderably more difficult to work with because they have highmolecular weights, are polydisperse, organic insoluble, pH andtemperature sensitive, and produce viscous solutions even at diluteconcentrations. Thus, it is a challenge to create tissue engineeredscaffolds with biomimetic porous networks in HA and SA hydrogels. Toaddress this problem the present invention describes acrystal-templating technique that uses in situ crystallization to carveout pores within biopolymer hydrogels. Salts and small organic moleculescan precipitate as crystalline branching networks under certainconditions. The similarity between such networks and the dendriticpatterns of microvasculature and neuronal dendritic trees prompted us touse in situ crystallization to template biopolymer hydrogels withmacroporous networks. SA and HA are both linear, unbranched, anionic,high molecular weight polysaccharides (their chemical structures aredepicted in FIG. 1), however they are distinguished by their mode ofcrosslinking HA can be crosslinked by derivatization with methacrylateside chains and exposure to UV light and a photoinitiator [25]. Alginatecan be crosslinked by addition of divalent cations which crosslinkadjacent guluronic acid blocks [24]. Both modes of crosslinking arecompatible with the crystal-templating technique of the presentinvention. Urea was chosen as the crystallite because of its high watersolubility and propensity for extensive hydrogen bonding to permitinteraction with biopolymer chains in solution.

The method described in the present invention includes five steps: filmcasting, solvent evaporation, crystal growth, crosslinking, and rinsing.These steps are depicted in FIG. 1. Small droplets (˜2 μL) of biopolymerand urea were cast onto microscope slides. The droplets were evaporatedat ˜50% relative humidity to produce thin hydrated films of about 5 mmin diameter on microscope slides. Solvent evaporation is required toachieve the super-saturation conditions necessary for crystallization.Evaporation also greatly increases the biopolymer concentration andsolution viscosity. The combination of high viscosity and hydrogenbonding suppresses spontaneous urea crystallization and facilitatessuper-saturation. Urea seed crystals were deposited on the tips of afine pair of tweezers and applied to the centers of each HA/urea andSA/urea film. Crystal growth began immediately and produced longdendritic branches that extended from the center to the edge of thefilm. Within seconds the entire volume of the hydrogel films were filledwith urea crystals. These crystals comprised the urea crystal template.

FIG. 1 shows the chemical structures of biopolymers used in thesehydrogels and schematic of the crystal-templating technique. Crystaltemplated hydrogels were created by casting solution of biopolymer andurea, e.g., hyaluronic acid and alginate. The solvent was evaporated toachieve supersaturation conditions concentrations of urea. As seen in 1the biopolymer-urea droplet is added to the glass slide, followed bydrying 2. Resulting in the application of a seed crystal to the centerof the film nucleated crystallization as seen in 3. Followingcrystallization the biopolymer is crosslinked by either UV or calcium 4.A water rinse 5 removes the urea crystals leaving behind a macroporoushydrogel templated 6 with the pattern of the urea crystals.

After the completion of the crystallization the films were crosslinkedby either UV exposure or calcium as appropriate. Both methods ofcrosslinking are rapid and accurately preserve the configuration of thecrystal template. After crosslinking the crystals were easily removedwith water. The end products were hydrogels with dendritic macroporousnetworks. Crystal-templated HA hydrogels are shown in FIG. 2. These thinfilms are easily observable by transmission light microscopy. Dropletscontaining both biopolymer and urea crystals (i.e., biopolymer-urea“composites”) were highly birefringent when observed through crossedpolarizers. These composite films exhibited a characteristic maltesecross pattern indicative of radial alignment. The colors of thecomposites in polarized light appeared to correspond to film thickness;the films were thickest at the center and thinned out toward the edges.After crosslinking and rinsing, the hydrogels lost most of theirbirefringence and exhibited only a faint white color when viewed throughcrossed polarizers. The hydrogel morphology consisted of alignedstraight “fibrils” that were the inverse shape of the crystal template.

FIGS. 2A to 2F are polarized light (FIGS. 2A and 2D) and phase contrast(FIGS. 2B, 2C, 2E, and 2F) microscopy images of crystal templated HAhydrogel. FIGS. 2A to 2D are images of the uncrosslinkedhydrogel-crystal composite. FIGS. 2E and 2F images of the hydrogelsafter crosslinking and removal of the urea crystals. Crystal growth wasnucleated at the center of each hydrogel and grew radially outwardtoward the edge of the hydrogel. The hydrogel-crystal composites in thefirst column exhibit a maltese cross characteristic of alignment. Thecrystal template pattern was retained by the hydrogel after crosslinkingand rinsing. Scale bars are 1000 μm in FIGS. 2A to 2C and 100 μm inFIGS. 2D to 2F.

Images of urea crystal growth were captured using video microscopy. FIG.3A depicts high magnification images of a growing urea crystal front.FIGS. 3A to 3C are high magnification images of urea crystals andtemplated hyaluronic acid hydrogels. FIG. 3A is a time lapse series ofthe urea crystal front captured with video Brightfield microscopy.Images are five seconds apart. Scale bar=10 μm. FIG. 3B is an AFM imageof the surface of a rinsed urea-templated HA hydrogel in air. The ridgescorrespond to the HA hydrogel. The grooves between the ridges hadcontained urea crystals that were washed out. This image demonstratesthat HA hydrogel was templated by the crystallization of urea. FIG. 3Cshows a profile of the AFM image perpendicular to the orientation of theridges.

It was found that the crystals were thin tightly packed needles thatsprouted a high density of branches. As the urea branches approachedneighboring crystals the local concentration of urea became depleted andbranch growth was terminated; therefore, the longest continuous brancheswere those that grew in the most radial direction away from the point ofnucleation. Direct observation of crystal growth confirmed that thecrystals grew continuously; therefore, we conclude that the porousnetwork within the crystal-templated hydrogels is also continuous.

Branches were not observed when urea crystals were grown in the absenceof biopolymer which indicates the importance of viscosity to inductionof dendritic growth. Such an effect has been observed by others thathave examined crystal growth in the presence of polymers [26, 27]. Wefound that the growth rate was highly dependent on the ratio of urea toHA concentrations. This ratio was adjusted by fixing the HAconcentration at 10 mg/mL and varying the urea concentration from 2.5 to180 mg/mL. Crystal growth could not be nucleated for a ratio of 0.25.For increasing ratios, the growth rate also increased and reached a rateof 1000 μm/sec for a ratio of 6. For ratios of 8 and greater a viscousliquid was expelled from the crystals shortly after crystallization.This liquid was easily visible under polarized light as dark liquid onthe surface of the birefringent urea crystals. The liquid was likelyrich in HA and photoinitiator which are impurities with respect to ureacrystals. At higher ratios the urea crystals became increasingly moretightly packed and less branched. Bulk polyhedral urea crystals wereobserved on the surface of the needle crystals when the ratio was 18. Weconcluded that ratios of 6 and under were most effective at templatingthe biopolymer because this ensured that the hydrogel was templated bythe crystals rather than excluded from the template.

The micro-topography of a rinsed urea-templated HA hydrogel was observedby contact mode AFM in air (FIG. 3B). The surface of the hydrogelconsisted of alternating parallel valleys and ridges. The valleys werecarved out by the growth of urea crystals. During crystallization, thecrystal front expels the biopolymer as an impurity. Thus, the biopolymerwas compressed into the interstices between urea crystals and formed theradially aligned thin ridges. A profile of the hydrogel perpendicular tothe length of the ridges is shown in FIG. 3C. The dimensions of theseridges, less than 1 μm in height and spaced between 1 and 2 μm apart,are consistent with our observations under the light microscope in FIG.3A.

Urea crystal growth can be nucleated two ways. Spontaneous nucleationoccurs when the concentration of urea exceeds a criticalsuper-saturation. Typically, spontaneous nucleation occurred on theedges of the hydrogels. Hydrogels never had more than one spontaneousnucleation event because crystal growth was very rapid.

FIGS. 4A to 4C show that the urea seed crystal technique can be used tocontrol the morphology of crystals and pores within HA hydrogels. FIG.4A shows three separate nucleation points produced a hydrogel with threeradial patterns. FIG. 4B shows a line of nucleation points produced ahydrogel with linear alignment of crystals. FIG. 4C shows a magnifiedimage of FIG. 4B. Scale bars are 1000 μm (FIG. 4A and FIG. 4B) and 100μm (FIG. 4C).

Applying a seed crystal to initiate urea crystal growth was a simplemethod for controlling the macro-morphology structure of the crystaltemplate. This was done in conditions that suppressed spontaneousnucleation by partially drying the hydrogel droplets under humidconditions. At equilibrium under humid conditions the hydrated filmshave a concentration of urea great enough to sustain crystal growth buttoo low for spontaneous nucleation. Un-nucleated hydrogel films could bemaintained for days until the introduction of a seed crystal. Thispermits selection of both the time and location of the nucleation. Filmsproduced by the application of one seed in the center of the film aredepicted in FIG. 2. Application of three seeds produced the three domaintemplate in FIG. 4A. Individual seeds produced radial crystal growth,whereas a line of seeds produced parallel alignment throughout thehydrogel film (FIG. 4B). Thus, seed nucleation is a simple method forengineering template morphology.

The droplets deposited on microscope slides formed thin films a fewmicrons thick as estimated by electron microscopy. Crosslinked filmscould be released from the surfaces of the microscope slides byagitation and transferred to solution for storage if desired. Thesefilms remained intact and did not fall apart, but could beenzymmatically degraded by hyaluronidase and dissolved by treatment witha calcium chelator, EDTA. Scale-up of the urea-templating procedure wasaccomplished for both HA and SA using a procedure similar to that usedfor the droplets. The scaled-up films were prepared by casting 2.6 mL ofsolution into 12-well plates with diameters of 2.2 cm per well. Three tofour days were required to evaporate solvent. Crystal growth could benucleated both spontaneously and by seed crystal.

FIGS. 5A to 5F shows the process of urea-templating was scaled up fromdroplets to thick films. FIG. 5A is a photograph of scaled-upfreestanding films of plain and urea-templated HA and alginate. FIGS. 5Bto 5E are SEM images of crystal templated HA after crosslinking andrinsing. FIG. 5B is a top down view. HA hydrogel was sculpted into a“fibrous” morphology. In FIG. 5C, this portion of the film was curledback to expose the crevices in the hydrogel that had been created byurea crystals. FIG. 5D shows a cross-section of the hydrogelperpendicular to the pore orientation depicting that the pores arepresent throughout the thickness of the hydrogel. FIG. 5E is a parallelcross-section depicting the fibrillar morphology. FIG. 5F is a magnifiedview of FIG. 5E.

Crystal-templated films were opaque white and had a fibrillar morphologyobservable even by eye (FIG. 5A). In contrast, non-templated films of HAwere transparent and featureless. Non-templated SA films were a lightmilky white and had a fine granular morphology when viewed under phasecontrast microscopy. All templated and non-templated HA and SA filmswere durable, pliable and easy to handle in both dry and swollenconditions.

Scaled-up templated films were too thick for their features to beobserved by optical microscopy; therefore, the morphology of these filmswas investigated by SEM. Dehydration of swollen hydrogel specimens caneasily create artifacts in the hydrogel structure and particularly onthe surface. To minimize such artifacts the SEM specimens wereextensively soaked and rinsed with methanol rather than water to removeurea. Urea is highly soluble in methanol (˜160 mg/mL) and the templatedhydrogel swelled minimally. The urea crystals had been so tightly packedthat the templated HA hydrogel had a fibrillar appearance. When observedfrom the top-down a repeating “arrowhead” fibrillar morphology of thefilm was clearly revealed (FIG. 5B). A curled portion of the film viewedat an angle revealed the interior crevices among the fibers that hadcontained the crystal template (FIG. 5C). Cross-sections of the filmperpendicular and parallel to the fiber orientation are depicted inFIGS. 5D and 5E, respectively. These cross-sections demonstrate that thepore and fiber morphology was present throughout the thickness of thefilm and not confined to the surfaces; thus, confirming that thetemplates are three-dimensional.

The crystal-templating technique is different from existing technologybecause the crystals are grown within the hydrogel. That is,crystallization begins at a point of nucleation and then radiatesoutwards filling the entire three dimensional volume. This process ofgrowth ensures that the resulting pores are interconnected and oriented.Importantly, the crystals are suspended within the viscous biopolymersolution permitting crystal growth in three dimensions. Scale-up can beachieved because crystallization occurs rapidly over large distances.Crystallization of urea within HA and SA hydrogels was both easilyreproducible and highly robust with respect to the concentrations ofurea and biopolymer. This robustness to concentration permitted thehydrogels to be scaled from droplets to large films. The formation ofthe crystal templates was, however, sensitive to ambient humidity whichaffected the rate of solvent evaporation and the final water content ofthe evaporated, equilibrated hydrogel films. This sensitivity wascircumvented through the use of controlled humidity conditions. Althoughcrystal-templating lacks precise control over the final pore morphologythis can be alleviated through temporal and spatial control of thenucleation event through the use of seed nucleation.

In addition to hyaluronic acid and alginate poly(ethylene glycol)acrylate hydrogels are also patternable by urea crystallization.Exploration of other crystallites and crystal growth conditions willyield a range of template morphologies. For example, engineeredpotassium phosphate templates were determined that possess dendriticstructures that are much larger and thicker than the urea templates.Crystal engineering is a set of techniques that tailors thesupramolecular assembly of crystalline materials by manipulating crystalgrowth conditions. Important parameters are the concentrations andratios of biopolymer and urea, solution viscosity, pH, and temperature.Additives such as surfactants and chiral molecules can selectivelyadhere to crystal faces and thereby control the branching, size andchirality of the resulting crystal structures [28]. Such techniques maybe able to refine the crystal structures and to produce a wide selectionof crystal templates.

The applications of crystal-templated hydrogels extend beyond tissueengineering because these hydrogels present a unique platform for thecreation of composite materials. For example, infusion of the pores withcell adhesive proteins would be ideal for tissue engineering scaffolds.Polymerization reactions and biomineralization within the pores canyield novel composites in which one component is distributed throughoutthe other component in oriented dendritic micron-sized pores.

Sodium hyaluronate from Streptococcus equi of molecular weight 1.6×10⁶Da as indicated by supplier and low viscosity alginic acid from brownalgae were obtained from Sigma-Aldrich (St. Louis, Mo.).Tetra-functional poly(ethylene glycol) acrylate (PEG4A, MW=10 kDa) wasobtained from SunBio. Photoinitiator, Irgacure 2959, was obtained fromCiba Specialty Chemicals (Basel, Switzerland). Photopolymerizations wereinitiated by a longwave UV lamp filtered around 365 nm and with anintensity of 22 mW/cm2 (Blak-Ray B-100A, UVP, Upland, Calif.).

Photocrosslinkable HA was prepared by our standard procedure ofderivatization of HA with glycidyl methacrylate to yield GMHA [25].Urea-templated GMHA films were prepared as depicted in FIG. 1A. Anaqueous solution was prepared of 10 mg/mL GMHA with 40 mg/mL urea and0.5 mg/mL photoinitiator (Irgacure 2959) in distilled deionized water.Droplets of 2 μL were dispensed onto a glass microscope slide. Thesolvent was partially evaporated overnight at ambient conditions or insealed containers with ˜50% relative humidity maintained by saturatedsolutions of calcium nitrate. GMHA is very hygroscopic and retainedresidual moisture in equilibrium with the humid atmosphere. Thus,evaporation yielded viscous hydrogel films that were super-saturatedwith urea. After drying, urea seed crystals were deposited onto the tipsof a fine pair of tweezers by scraping the tweezers against solid urea.Then the tweezers were carefully touched to the center of each dropletto nucleate crystallization. Similarly, a razor-blade scraped againsturea was used to nucleate straight lines. GMHA was crosslinked by 1minute of exposure to UV. The hydrogels were rinsed in water to removeurea.

Templated alginate hydrogels were prepared as shown in FIG. 1B. Aqueoussolutions of 10 mg/mL alginate with 40 mg/mL urea were dispensed ontomicroscope slides and air-dried. After crystallization the hydrogelswere crosslinked by covering the droplet 200 mg/mL calcium chloridesolution for one minute. Rinsing with water removed excess calciumchloride and urea.

Templated PEG4A hydrogels were prepared from aqueous solutions of 60mg/mL PEG4A, 40 mg/mL urea and 0.5 mg/mL Irgacure 2959. Potassiumphosphate templated GMHA was prepared from a solution of 20 mg/mL GMHA,10 mg/mL potassium dihydrogen phosphate and 0.5 mg/mL Irgacure 2959.These droplets were incubated in humid chambers equilibrated withsaturated sodium chloride solutions until crystal growth was complete.

Scale-up of Crystal Templated Hydrogels. Thick crystal-templatedhydrogels were prepared under sterile conditions to preventcontamination during solvent evaporation. Aqueous solutions wereprepared as described above, filter sterilized (0.22 μm PVDF,Millipore), and dispensed into sterile, non-tissue culture treated12-well plates. Each well was 2.2 cm in diameter and a volume of 2.6 mLof sterile solution was dispensed per well. The solvent was evaporatedin a sterile horizontal flow hood in the dark for four days. During thistime urea crystallization either nucleated spontaneously or wasnucleated by the seed crystal technique. Once nucleated, crystallizationis rapid and therefore spontaneously nucleated films had only onenucleation point. GMHA films were crosslinked by 1 minute of UVexposure, and rinsed extensively with exchanges of water over severaldays to remove urea. Alginate films were crosslinked by immersion in 200mg/mL calcium chloride for twenty minutes and then rinsed extensivelywith water.

Plain GMHA films required an additional humidifying step afterair-drying and before photocrosslinking This step was required becausethe photocrosslinking reaction was moisture sensitive. It is likelynecessary for water to be retained within the film to permit diffusionof photoinitiator and movement of GMHA chains during photoexposure.Therefore, air-dried films were placed in a sealed container at ˜85%relative humidity which was achieved by equilibration with saturatedpotassium chloride solution. GMHA films were incubated under theseconditions for four days and then photocrosslinked by 1 minute ofexposure to UV.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

While the present invention has been described with respect to a limitednumber of embodiments, those skilled in the art will appreciate numerousmodifications and variations therefrom. It is intended that the appendedclaims cover all such modifications and variations as fall within thetrue spirit and scope of this present invention.

What is claimed is:
 1. A hydrogel film comprising: first and secondopposing ends; chitosan; urea; and at least one crystal; wherein the atleast one crystal is (a) dendritically branched, (b) at least 1 micronin diameter, and (c) comprises the urea.
 2. The hydrogel film of claim1, wherein the hydrogel film has top and bottom surfaces connecting thefirst and second ends, and the at least one crystal is distributed fromthe top surface to the bottom surface and from the first end to thesecond end.
 3. The hydrogel film of claim 1, wherein: the at least onecrystal forms a continuous crystal network, connecting the first andsecond ends, including first and second crystal portions; and thehydrogel film further comprises a compressed fiber, including a ridge,compressed between the first and second crystal portions.
 4. Thehydrogel film of claim 3, wherein the compressed fiber is included in aplurality of compressed fibers formed among alternating valleys andridges.
 5. The hydrogel film of claim 1, wherein the at least onecrystal forms a continuous crystal network.